| Co-Investigator(Kenkyū-buntansha) |
HIRASAWA Toshiko The same institution and department Technical Associate, 化学研究所, 教務職員
YOKOIGAWA Kumio The same institution and department Research Associate, 化学研究所, 助手 (60230637)
ESAKI Nobuyoshi The same institution and department Associate Professor, 化学研究所, 助教授 (50135597)
吉村 徹 京都大学化学研究所, 助手 (70182821)
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| Budget Amount *help |
¥6,300,000 (Direct Cost: ¥6,300,000)
Fiscal Year 1991: ¥2,900,000 (Direct Cost: ¥2,900,000)
Fiscal Year 1990: ¥3,400,000 (Direct Cost: ¥3,400,000)
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| Research Abstract |
The chromosomal DNA of mouse GSHPx gene was reconstructed to express in E. coli. It contains a single intron within the structural gene. Then, BamHI sites were introduced at both the ends of the intron by oligonucleotide-directed mutagenesis, and the following digestion with BamHI and ligation eliminated the intron. The reconstructed gene was subcloned into the E. coli expression vector, pKK223-3, and E. coli JM109 cells were transformed with the resulting plasmid. The transfomant was incubated with ^<35>S-methionine and ^<75>Se-Na_2SeO_3 to label the proteins. However, GSHPx was not synthesized under the conditions used.
The gene fragments of GSHPx which contain the TGA codon encoding the SeCys residue were fused with the gene of beta-gal. When the TGA is readthrough, the fused protein most probably has beta-gal activity, which is easily detected with X-Gal. The transformants showed beta-gal activity only when cultured anaerobically. The fused proteins produced under anaerobic conditio
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ns were labeled with ^<75>Se-Na_2SeO_3. These results suggest that some factors suppressing the nonsense codon TGA are formed under anaerobic conditions, and consequently selenium is incorporated into the fused protein.
We synthesized a selenocysteine-containing peptide, metalloselenonein, which contains selenocysteine residues substituted for all cysteine residues in Neurospora crassa copper metallothionein, using an automated peptide synthesizer. Metalloselenonein binds 3 gram atoms of Cu (I) per mol. This adduct showed a broad absorption band between 230 and 400 nm and a fluorescence band at 395 nm, which can be attributed to copper-selenolate coordination. The circular dichroism spectrum of the Cu-metalloselenonein complex showed a positive CD band around 245 nm attributable to asymmetry in metal coordination. The Cu-bound metalloselenonein was analyzed by Cu-and Se-EXAFS. the results revealed that three different Cu-Se bonds occur in Cu-SeMT, and the ratio of occurrence was 3, 1, and 3, respectively. Less
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